Mixed beams of ionizing radiation in our environment originate from space, the bedrock and our own houses. Radiotherapy patients treated with boron neutron capture therapy or with high energy photons are also exposed to mixed beams of gamma radiation and neutrons. Earlier investigations have reported additivity as well as synergism (a greater than additive response) when combining radiations of different linear energy transfer. However, the outcome seemed to be dependent on the experimental setup, especially the order of irradiation and the temperature at exposure. A unique facility allowing simultaneously exposure of cells to X-rays and 241Am alpha particles at 37 ºC was constructed and characterized at the Stockholm University (Paper I). To investigate the cytogenetic response to mixed beam irradiation (graded doses of alpha particles, X-rays or a mixture of both) several different cell types were utilized. AA8 Chinese Hamster Ovary cells were analyzed for clonogenic survival (Paper I), human peripheral blood lymphocytes were analyzed for micronuclei and chromosomal aberrations (Paper II and Paper III respectively) and VH10 normal human fibroblasts were scored for gamma-H2AX foci (Paper IV). For clonogenic survival, mixed beam results were additive, while a significant synergistic effect was observed for micronuclei and chromosomal aberrations. The micronuclei dose responses were linear, and a significant synergistic effect was present at all investigated doses. From the analysis of micronuclei distributions we speculated that the synergistic effect was due to an impaired repair of X-ray induced DNA damage, a conclusion that was supported by chromosomal aberration results. Gamma-H2AX foci dose responses were additive 1 h after exposure, but the kinetics indicated that the presence of low LET-induced damage engages the DNA repair machinery, leading to a delayed repair of the more complex DNA damage induced by alpha particles. These conclusions are not necessary contradictory since fast repair does not necessarily equal correct repair. Taken together, the observed synergistic effects indicate that the risks of stochastic effects from mixed beam exposure may be higher than expected from adding the individual dose components. / <p>At the time of the doctoral defence the following papers were unpublished and had a status as follows: Paper nr 3: Manuscript; Paper nr 4: Manuscript.</p> / DNA damage and repair in cells exposed to mixed beams of radiation

<p>Efficient and correct repair of DNA damage, especially DNA double-strand breaks (DSBs), is vital for the survival of individual cells and organisms. Defects in the DNA repair may lead to cell death or genomic instability and development of cancer. </p><p>The repair of DSBs in cell lines with different DSB rejoining capabilities was studied after exposure to ionising radiation. A new cell lysis protocol performed at 0ºC, which prevents the inclusion of non-true DSBs in the quantification of DSBs by pulsed-field gel electrophoresis (PFGE), was developed. Results showed that when the standard protocol at 50ºC was used, 30-40% of the initial yield of DSBs corresponds to artifactual DSBs. The lesions transformed to DSBs during incubation at 50ºC were repaired within 60-90 minutes <i>in vivo</i> and the repair was independent of DNA-PK, XRCC1 and PARP-1.</p><p>Non-homologous end-joining (NHEJ) is the major DSB repair pathway in mammalian cells. We show that DSBs are processed into long single-stranded DNA (ssDNA) ends after ≥1 h of repair in NHEJ deficient cells. The ssDNA was formed outside of the G₁ phase of the cell cycle and only in the absence of the NHEJ proteins DNA-PK and DNA Ligase IV/XRCC4. The generation of ssDNA had great influence on the quantification of DSBs by PFGE. The standard protocol caused hybridisation of the ssDNA ends, resulting in overestimation of the DSB repair capability in NHEJ deficient cells.</p><p>DSBs were also quantified by detection of phosphorylated H2AX (γ-H2AX) foci. A large number of γ-H2AX foci still remaining after 21 h of repair in an NHEJ deficient cell line confirmed the low repair capability determined by PFGE. Furthermore, in normal cells difficulty in repairing clustered breaks was observed as a large fraction of γ-H2AX foci remaining 24 h after irradiation with high-LET ions.</p>

Efficient and correct repair of DNA damage, especially DNA double-strand breaks (DSBs), is vital for the survival of individual cells and organisms. Defects in the DNA repair may lead to cell death or genomic instability and development of cancer. The repair of DSBs in cell lines with different DSB rejoining capabilities was studied after exposure to ionising radiation. A new cell lysis protocol performed at 0ºC, which prevents the inclusion of non-true DSBs in the quantification of DSBs by pulsed-field gel electrophoresis (PFGE), was developed. Results showed that when the standard protocol at 50ºC was used, 30-40% of the initial yield of DSBs corresponds to artifactual DSBs. The lesions transformed to DSBs during incubation at 50ºC were repaired within 60-90 minutes in vivo and the repair was independent of DNA-PK, XRCC1 and PARP-1. Non-homologous end-joining (NHEJ) is the major DSB repair pathway in mammalian cells. We show that DSBs are processed into long single-stranded DNA (ssDNA) ends after ≥1 h of repair in NHEJ deficient cells. The ssDNA was formed outside of the G1 phase of the cell cycle and only in the absence of the NHEJ proteins DNA-PK and DNA Ligase IV/XRCC4. The generation of ssDNA had great influence on the quantification of DSBs by PFGE. The standard protocol caused hybridisation of the ssDNA ends, resulting in overestimation of the DSB repair capability in NHEJ deficient cells. DSBs were also quantified by detection of phosphorylated H2AX (γ-H2AX) foci. A large number of γ-H2AX foci still remaining after 21 h of repair in an NHEJ deficient cell line confirmed the low repair capability determined by PFGE. Furthermore, in normal cells difficulty in repairing clustered breaks was observed as a large fraction of γ-H2AX foci remaining 24 h after irradiation with high-LET ions.

Ces dernières années, le paradigme de la radiobiologie selon lequel les effets biologiques des rayonnements ionisants ne concernent strictement que les dommages à l'ADN et les conséquences liées à leur non réparation ou à leur réparation défectueuse, a été remis en question. Ainsi, plusieurs études suggèrent que des mécanismes «non centrés » sur l'ADN ont une importance significative dans les réponses radio-induites. Ces effets doivent donc être identifiés et caractérisés afin d’évaluer leurs contributions respectives dans des phénomènes tels que la radiorésistance, les risques associés au développement de cancers radio-induits, les conséquences des expositions aux faibles doses. Pour ce faire, il est nécessaire : (i) d'analyser la contribution de ces différentes voies de signalisation et réparation induites en fonction de la dose et de la zone d’irradiation; (ii) d’’étudier les réponses radio-induites suite à l’irradiation exclusive de compartiments subcellulaires spécifiques (exclure les dommages spécifiques à l'ADN nucléaire); (iii) d’améliorer la connaissance des mécanismes moléculaires impliqués dans les phénomènes de radiosensibilité/radiorésistance dans la perspective d’optimiser les protocoles de radiothérapie et d’évaluer in vitro de nouvelles thérapies associant par exemple les effets des rayonnements ionisants et de nanoparticules d’oxydes métalliques. Les microfaisceaux de particules chargées offrent des caractéristiques uniques pour répondre à ces questions en permettant (i) des irradiations sélectives et en dose contrôlée de populations cellulaires et donc l’étude in vitro des effets « ciblés » et « non ciblés » à l'échelle cellulaire et subcellulaire, (ii) de caractériser l’homéostasie de cultures cellulaires en réponses à des expositions aux rayonnements ionisants et/ou aux nanoparticules d’oxydes métalliques (micro-analyse chimique multi-élémentaire). Ainsi, au cours de ma thèse, j'ai validé et exploité des méthodes d’évaluation qualitatives et quantitatives (i) in cellulo et en temps réel de la réponse radio-induite de compartiments biologiques spécifiques (ADN, mitochondrie, …) ; (ii) in vitro de la radiosensibilité de lignées sarcomateuses issues de patients; et (iii) in vitro des effets induits par des expositions à des nanoparticules d'oxydes métalliques afin d’évaluer leur potentiel thérapeutique et anti-cancéreux. / Few years ago, the paradigm of radiation biology was that the biological effects of ionizing radiations occurred only if cell nuclei were hit, and that cell death/dysfunction was strictly due to unrepaired/misrepaired DNA. Now, next this “DNA-centric” view several results have shown the importance of “non-DNA centered” effects. Both non-targeted effects and DNA-targeted effects induced by ionizing radiations need to be clarified for the evaluation of the associated radiation resistance phenomena and cancer risks. A complete overview on radiation induced effects requires the study of several points: (i) analyzing the contribution of different signaling and repair pathways activated in response to radiation-induced injuries; (ii) elucidating non-targeted effects to explain cellular mechanisms induced in cellular compartments different from DNA; and (iii) improving the knowledge of sensitivity/resistance molecular mechanisms to adapt, improve and optimize the radiation treatment protocols combining ionizing radiations and nanoparticles. Charged particle microbeams provide unique features to answer these challenge questions by (i) studying in vitro both targeted and non-targeted radiation responses at the cellular scale, (ii) performing dose-controlled irradiations on a cellular populations and (iii) quantifying the chemical element distribution in single cells after exposure to ionizing radiations or nanoparticles. By using this tool, I had the opportunity to (i) use an original micro-irradiation setup based on charged particles microbeam (AIFIRA) with which the delivered particles are controlled in time, amount and space to validate in vitro methodological approaches for assessing the radiation sensitivity of different biological compartments (DNA and cytoplasm); (ii) assess the radiation sensitivity of a collection of cancerous cell lines derived from patients in the context of radiation therapy; (iii) study metal oxide nanoparticles effects in cells in order to understand the potential of nanoparticles in emerging cancer therapeutic approaches.